Back at the bench (1998 on), we were setting up the colony of X. tropicalis in the lab. Our colony was founded with animals from the
Grainger lab at UVa;
this lab maintains a large, inbred population in specialized quarters, using
well water etc. One of our contributions to the tropicalis consortium has been to develop a system for reliably and rapidly rearing tropicalis with minimal expense (see
Kelley lab web page).

In order to tap into the experimental advantages of tropicalis we first needed
to explore its vocal system. We examined the larynges of males and females
and recorded from muscle fibers, we cloned several genes (including the tropicalis estrogen receptor; Wu et al., 2003a) and we began to characterize their vocal
behaviors. None of the calls that we recorded matched the tropicalis calls
recorded by Loumont in Africa (Kobel et al., 1996) and we began to suspect
that there might be more than one population of 20/40 chromosome Xenopodinae.
The recordings of tropicalis songs also sharpened our interest in the evolution
of song in this group (see XENOPUS SONGS; GENETICS AND EVOLUTION) and we began
to think about visiting other parts of Africa to collect frogs and study songs.
In particular we were interested in the effect of differences in ploidy on
songs and tropicalis/epitropicalis seemed like a good system to begin with.

One or another species in this pair had been
collected in a number of countries in Central Africa (reviewed in Tinsley,
1996) but many of these countries
did not seem especially suitable for biological studies because of political
instability. Christine Loumont very kindly gave me detailed advice about visiting
Cameroon, as Xenopus has been collected very extensively there (the
country is regarded as " the most important area for Xenopus species
diversity",
Tinsley et al., 1996), We thought, however, that we might be able to make a
novel contribution by focusing elsewhere within this region.

We went to Gabon. A major element in our choice
was a paper that Carl Hopkins delivered (at a symposium in honor of Peter
Marler's contributions
to animal communication in 1997) on his studies of signal evolution in electric
communication in African mormyrid fish. Carl has been collecting and examining
various species of these fish, focusing on the Ivindo River in Gabon, for more
than 30 years. Many of the new species that he has found cannot be distinguished
morphologically but can be told apart by the form of their electric organ discharge
(and now using molecular phylogenetic methods as well; see Hopkins, 1999).
I began to wonder whether there might not also be cryptic species of Xenopus and
whether we could distinguish these using their songs.

Figure 12 Sites for current field studies on Xenopus in this laboratory.

Attention to conservation is an essential obligation
of anyone interested in ecology and evolution and an important focus as well
for the student of animal behavior. Conservation decisions require knowledge
of the diversity of species so that appropriate choices can be made with
respect to development and to habitat preservation. Attention usually focuses
on charismatic mammal and bird species. Work on mormyrid fish, however, suggests
that small, insignificant -looking aquatic species can also make very important
contributions (http://www.nbb.cornell.edu/neurobio/hopkins/mkingsley.html).
The Mormyriformes are literally "poster children" (courtesy of Elf) in Gabon.
Could we add amphibians (with many species currently in decline), particularly
aquatic amphibians such as Xenopus, to biodiversity efforts and, at the same time, learn something about how their songs participate in species diversification?

These thoughts prompted a trip to Gabon in the
summer of 2001. The entire team on amphibian biodiversity includes Chris
Raxsworthy at the American Museum of Natural History, Michael Klemens of
the Wildlife Conservation Society, Ben Evans of Columbia's Center for Environmental
Research and Conservation, Martha Tobias and me. A planning grant from CERC and funds
from Columbia's Digital Knowledge Ventures, in connection with an electronic seminar on the neurobiology of animal communication, supported this first foray (which would not have been possible without considerable help from our collaborators and advice from C. Hopkins and John Sullivan at the AMNH, among many others).

Figure 13. Darcy and Martha prepare for Gabon!

We arrived in Libreville at the beginning of
August and joined forces with two of Carl's students, Matthew Arnegard and
Tim Uschold, in making contact
with Gabonese officials to secure the necessary permits to carry out our research
and for the exportation of biological materials. Olivier Pauwels and Sebastien
Evoue, who were carrying out a survey of the biodiversity of Gabonese herpetofauna
under the direction of Andre Kamden of the World Wildlife Fund on behalf of
Gabonese conservation efforts, were extremely helpful with respect to both
science and logistics.

Figure 14. At the World Wildlife Fund in Libreville,
Gabon

M. Samuel Ikogou, Directeur des Etudes at the Ecole
National des Eaux et Forets, had offered to host our field studies at Cap
Esterias, about 20km North of Libreville.

M. Jean Daniel Mbega of the Institut de Recherches Agronomiques et Forestieres
assisted this project in many ways including bringing us together with a student
at the ENEF, Marie-Agathe Manga Moubele, who carried out a project with us
at Cap Esterias Our first goal was to determine if any of the local ponds contained
Xenopus and we made and baited funnel traps, a bit smaller than those we used
in South Africa and floated them in the ponds. No frogs. A walk along the river
bank yielded a fish trap but no frogs.

Figure 15 At Cap Esterias, North of Libreville

Walking back from the river we met Florence who arranged to have fish traps
(les naces) made for us and also promised to catch us some frogs on Monday;
we crossed our fingers and headed in to Libreville.

The local market is held on the road between Libreville and Cap Esterias.
As it happens, Xenopus is regarded by the Gabonese as a particularly
tasty snack, especially rolled in leaves (en paquette) and poached in bouillon.
Laid out on a leaf in the market was our first Xenopus sighting.

Figure 16 Xenopus at the Saturday market en route
to Cap Esterias

Considerably cheered and relieved, we joined Carl
and his expedition and M. Mbega for a celebratory dinner at the Hotel Tropicana.

Figure
17 A farewell dinner at the Hotel Tropicana

A consultation with the Eaux et Forets faculty the next day painted a gloomier picture. It was the dry season and the frogs would be deep in the mud, very hard to dig out. We should come back in the rainy season, then we would be overrun with frogs. After a visit to the Chef du Cap to request permission, we went prospecting at the mouth of the Abaga. This scramble yielded mud puppies (Periopthalmus) but no frogs.

A consultation with the Eaux et Forets faculty the next day painted a gloomier
picture. It was the dry season and the frogs would be deep in the mud, very
hard to dig out. We should come back in the rainy season, then we would be
overrun with frogs. After a visit to the Chef du Cap to request permission,
we went prospecting at the mouth of the Abaga. This scramble yielded mud puppies
(Periopthalmus) but no frogs.

Figure 18 At the mouth of the Abaga.

Walking back from the village we stopped to chat with a woman who said that
her children had caught some frogs the previous day but they were all dead
now. Being sent off to catch more, the children returned with a bucket of small
Xenopus and we were in business.

During the dry season the local farms use water obtained from small wells
(puits), dug from the clayey soil. The frogs aestivate in the mud and when
digging breaks into their chambers they jump from the chambers into the water
where they are quite easily caught, usually by the children. The villagers
claim that, in fact, it is much easier to catch Xenopus this way during the
dry season than in the rainy season.

Figure 19 Catching Xenopus in Gabon.

Thus with considerable, and much appreciated, help
from the villagers of Cap Esterias, we were able to return to Columbia with
105 living frogs. We now
have a large (>100) breeding colony derived from these frogs. These frogs
are a polyploidy relative of Xenopus tropicalis called paratropicalis. They
were first described in a paper by Janina Tymkowska (1991) on karyotypes in
the genus; in that paper they are referred to as X. species nova VII.

During our trip to Gabon we were able to meet many Gabonese active in conservation
and in education and look forward to forming a co-operative alliance between
these efforts and our efforts at Columbia when we return (as we hope) in the
future.

More recently we have begun a collaborative study with Ben Evans, a former
lab member and fellow of Columbia's Earth Institute, on the evolution of the
Xenopodinae. Ben has reanalyzed the molecular phylogeny of the group using
mitochondrial and nuclear genes and has been able to pin point the origins
of the polyploidy species. Martha Tobias has been analyzing the advertisement
calls of all species in an attempt to track changes in vocal structure evolutionarily.
Ben has just returned from a trip to Cameroon where he found the dodecaploid
species longipes and we will be analyzing their calls as well.